The overall objective of this proposal is to explore the powers of the ocular proteomics approach for obtaining novel information on protein composition of individual cell types and their individual subcellular compartments. The Specific Aim is to generate a proteome map of the photoreceptor cell layer of the retina where a large number of individual proteins will be assigned to their relative subcellular locations. This will be achieved by conducting comprehensive mass spectrometry protein sequencing in progressive tangential sections throughout the photoreceptor layer. The data will be summarized in a database where all identified proteins will be grouped according to their subcellular localization, as well as their potential involvement in various functional groups. Obtaining such a proteome map would enable us and many other investigators to refine existing hypotheses and to formulate novel hypotheses regarding a broad array of photoreceptor functions and diseases. The technologies developed in this study will also serve as a template for similar investigations addressing protein distributions in other layers of the retina as well as other layered tissues. The project described in this application fulfills the goals of this Program Announcement (PAR-03-107) for two major reasons. First, it has a potential to lead to a significant technological breakthrough by establishing the methodologies for conducting large scale proteomics analyses of ocular tissues. As we are entering the post-genomics era, there is a pressing need to developing advanced proteomics approaches in many areas of ophthalmology and visual sciences. To our knowledge, several key aspects of the proposed studies (such as the analysis of serial tissue sections and the use of "two-dimensional chromatography" peptide separation protocols) have not been used in application to any ocular tissue. Second, this project aims to acquire a body of data that has a potentially high-impact on vision research because knowledge of the protein composition of individual photoreceptor compartments will benefit many scientists studying a broad array of problems in photoreceptor cell biology and elucidating molecular bases of eye diseases. [unreadable] [unreadable] [unreadable]